Natural products from organisms as diverse as bacteria, plants, and marine invertebrates constitute a rich source of molecules with wide-ranging bioactivities related to human disease, including antibiotics and anti- cancer agents. A plentitude of these structurally complex secondary metabolites are synthesized by large enzyme complexes, polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs), in a linear assembly-line manner. PKSs/NRPSs consist of multiple polypeptides (modules), each with multiple functional domains that covalently load appropriate building blocks (e.g., malonyl groups for PKSs and activated amino acids for NRPSs) and sequentially condense them onto the growing natural product chain. Often, additional enzymes are involved for further processing, such as attachment of carbohydrates. There is also enzymatic variety within each module such that, e.g., dehydration, reduction, and alkylation reactions may occur at any position in the growing natural product chain for increased structural diversity. Tremendous advances in our understanding of natural product biosynthetic pathways are beginning to allow pathway engineering for generation of compounds with new or improved bioactivities. However, in many cases, valuable natural products are known but the corresponding biosynthetic pathways remain undiscovered due to, e.g., challenges in genome sequencing. For such systems, pathway discovery at the protein rather than DNA level is emerging as an attractive approach that also verifies biosynthetic protein expression. However, due to the complexity of collected metaproteomic samples, targeted methods are needed. This proposal describes the development of innovative methods for targeted PKS/NRPS proteomics, as well as their application for pathway discovery in the dinoflagellate Karenia brevis. This marine plankton produces the highly structurally complex brevetoxins, responsible for the deaths and illnesses associated with the Florida red tide, as well as the antitoxin, brevenal, currently in clinical trials for treatment of asthma and cystic fibrosis. We will harness the high infrared absorption of phosphopantetheine (Ppant) prosthetic groups on PKSs/NRPSs to selectively detect Ppant-containing peptides in proteolytic digests with mass spectrometry in a parallel rather than the conventional sequential manner. This innovative strategy will be validated in highly complex metaproteomic samples such as the tunicate/microbial symbiont producer of the approved anti-cancer agent ET-743, for which we recently demonstrated feasibility of biosynthetic protein detection. We will also develop suitable bioinformatic approaches for automated mining of such complex datasets. For increased selectivity, we will develop IR-active chemical probes, resembling secondary metabolite biosynthetic intermediates, for loading onto PKSs/NRPSs. These approaches will be applied for PKS discovery in collected K. brevis samples. Biosynthetic pathway identification will allow characterization of the corresponding undoubtedly highly intricate biosynthetic mechanisms, and provide a gateway to sustainable drug production.